There is a very large demand for DNA analysis for a range of purposes and this has lead to the requirement for quick, safe, high throughput methods for the isolation and purification of DNA and other nucleic acids. Samples for use for DNA identification or analysis can be taken from a wide range of sources such as biological material such as animal and plant cells, faeces, tissue etc. also samples can be taken from soil, foodstuffs, water etc.
Existing methods for the extraction of DNA include the use of phenol/chloroform, salting out, the use of chaotropic salts and silica resins, the use of affinity resins, ion exchange chromatography and the use of magnetic beads. Methods are described in U.S. Pat. Nos. 5,057,426 and 4,923,978, EP 0 512 767 A and EP 0 515 484 A and WO 95/13368, WO 97/10331 and WO 96/18731. These patents and patent applications disclose methods of adsorbing nucleic acids on to a solid support and then isolating the nucleic acids. The previously used methods use some type of solvent to isolate the nucleic acids and these solvents are often flammable, combustible or toxic.
EP 0 707 077 A describes a synthetic water soluble polymer to precipitate nucleic acids at acid pH and release at alkaline pH. The re-dissolving of the nucleic acids is performed at extremes of pH, temperature and/or high salt concentrations where the nucleic acids, especially RNA, can become denatured, degraded or require further purification or adjustments before storage and analysis.
WO 96/09116 discloses mixed mode resins for recovering a target compound, especially a protein, from aqueous solution at high or low ionic strength, using changes in pH. The resins have a hydrophobic character at the pH of binding of the target compound and a hydrophilic and/or electrostatic character at the pH of desorption of the target compound.
WO 99/29703 and WO 02/48164 disclose the use of charge switch materials for purifying nucleic acid, binding nucleic acid in a sample to a solid phase at a low pH (e.g. pH 6) and releasing the nucleic acid at a higher pH (e.g. pH 8). WO 99/29703 exemplifies the use of solid phases incorporating histidine or polyhistidine groups, and WO 02/48164 further exemplifies the use of charge switch materials such as biological buffer, for example Bis-Tris.
There is also considerable current interest in methods of carrying active molecules such as DNA, drugs and other therapeutic agents and delivering them to a target site, particularly a target site in vivo.
Existing methods for delivering DNA to a target site include the delivery of DNA using modified retroviruses or adenoviruses, direct injection of naked DNA into the organism, or use of liposomes.
Synthetic delivery systems such as liposomes are advantageous over viruses for a number of reasons, including a reduced risk of immunogenic reaction, and the possibility of increased carrying capacity. However, for cationic liposomes, the positive charge on the surface of the delivery envelope can result in non-specific tissue uptake and non-specific interaction with negatively charged serum molecules, blood cells and the extra-cellular matrix. These interactions also sometimes cause precipitation. Anionic liposomes, on the other hand, achieve low encapsulation as a result of the inability of the DNA to interact with the coating liposome.
An alternative method for delivering DNA involves forming a complex between DNA and a polycation (Cotton 1993, Current Opinion in Biotechnology V4 p 705). U.S. Pat. No. 5,908,777 describes a method of forming a lipidic vector for delivery of therapeutic molecules which entails forming a complex between the desired substance and a polycation such as polylysine, and then mixing the complex with an anionic lipid preparation.
U.S. Pat. No. 5,679,559 describes a method for introducing DNA into cells, which involves providing a core of lipoprotein. This is associated with hydrophobic side chains of a positively charged biocompatible polymer, which in turn is associated with a nucleic acid molecule. Because the polymer is carried on the surface of the particle, the amount of polymer that can be carried by the particle is limited.
U.S. Pat. No. 6,383,811 discloses a delivery system in which a complex of DNA and polycation is associated with a negatively charged polymer, to render the particle as a whole negatively charged and thus to make delivery easier. The negatively charged polymer can be a separate polymer added to preformed DNA/polycation complexes, or it can be covalently bound to the polycation to form a polyampholyte, which is then complexed with the DNA. However, there is no disclosure that the polycation or polyanion should have charge switch properties, such that release of the DNA is induced by a change in the pH environment. There is also no disclosure that the components of the particle should be arranged in a multi-layer structure.